European Journal of Clinical Nutrition (2004) 58, 10–16 & 2004 Nature Publishing Group All rights reserved 0954-3007/04 $25.00 www.nature.com/ejcn ORIGINAL COMMUNICATION Effects of the interaction between the C677T 5,10-methylenetetrahydrofolate reductase polymorphism and serum B vitamins on homocysteine levels in pregnant women KN Kim1, YJ Kim2 and N Chang1* 1Department of Food and Nutritional Sciences, Ewha Womans University, Seoul, Korea; and 2Department of Obstetrics and Gynecology, Medical Research Center, Ewha Womans University, Seoul, Korea Objective: The purpose of this study was to investigate the effect of the interaction between the C677T mutation in the 5,10- methylenetetrahydrofolate reductase (MTHFR) genotypes and serum levels of B vitamins on serum homocysteine levels in pregnant women. Design: A cross-sectional study. Setting: Ewha Womans University Hospital, Seoul, Korea. Subjects: A total of 177 normal pregnant women, 24.671.1 weeks of gestation, in a 6-month period during 2001–2002. Interventions: Serum vitamin B2, vitamin B6, and homocysteine analyses were conducted using high-performance liquid chromatography methods. Serum folate and vitamin B12 concentrations were determined using a radioimmunoassay kit. MTHFR gene mutation was investigated by the polymerase chain reaction of a genomic DNA fragment. Results: Serum homocysteine was higher in women with the T/T genotype than those with the C/T or C/C genotype of the MTHFR gene (Po0.05). Serum homocysteine was negatively correlated with serum folate in all MTHFR genotypes (Po0.001), and the correlation between the two serum levels was the strongest in the T/T genotype. Serum homocysteine was higher in the subjects with the T/T MTHFR genotype only when the serum folate was below the median level. Explanatory power of B vitamin status as predictors of serum homocysteine levels was more pronounced in the T/T genotypes (68.5%) compared with the C/T (37.9%) or C/C genotypes (20.6%). Conclusions: Serum homocysteine levels in pregnant women varied significantly with MTHFR genotype and the serum B vitamin status. Higher serum folate, vitamin B2, and vitamin B12 concentrations may lessen the MTHFR genotypic effect on serum homocysteine levels. Sponsorship: This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (01-PJ1-PG1-01CH15-0009). European Journal of Clinical Nutrition (2004) 58, 10–16. doi:10.1038/sj.ejcn.1601729 Keywords: homocysteine; vitamin B2; folate; vitamin B12; MTHFR genotype; pregnancy *Correspondence: N Chang, Department of Food and Nutritional Introduction Sciences, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Korea. Elevated total serum homocysteine concentration in preg- E-mail: [email protected] nant women is an important issue as it is known to be related Guarantors: Ki Nam Kim, Namsoo Chang. to many adverse pregnancy outcomes including birth defects Contributors: NSC and YJK contributed to the design of the study, (Mill et al, 1995; Rosenquist et al, 1995), pre-eclampsia (Leeda subject recruiting, and manuscript preparation. KNK carried out the data analysis and the biochemical analyses of serum B vitamins and et al, 1998), placental abruption (Goddijn-Wessel et al, 1996), homocysteine. YJK was responsible for clinical data collection and the spontaneous abortion (Wouters et al, 1993), low birth weight analysis of the MTHFR polymorphism. NSC was the main person (Burke et al, 1992), and other maternal or fetal complications responsible for all stages of the study. All took part in the writing of (Aubard et al, 2000; Scholl & Johnson, 2000). the paper. Received 25 July 2002; revised 9 January 2003; Homocysteine is influenced by the nutritional status of accepted 13 January 2003 B vitamins (vitamin B2, vitamin B6, folate, and vitamin B12), MTHFR polymorphism and serum B vitamins in pregnant women KN Kim et al 11 whose metabolic steps are inter-related with each other vitamin B12 were calculated by CANPro II, a computerized (Perry, 1999; Ueland et al, 2001) and nutritional adequacy of nutrient intake assessment software program developed by these vitamins is essential to maintain the plasma homo- the Korean Nutrition Society (2002). cysteine levels within a normal homeostatic range. The Fasting venous blood was drawn from the antecubital vein. conversion step of tetrahydrofolate (THF) to 5,10-methylene Blood samples for vitamins and homocysteine were centri- THF is coupled with a PLP-dependent reaction and that of fuged for 10 min at 3000 Â g to separate the serum. The 5,10-methylene THF to 5-methyl THF, a FAD-dependent serum was divided into several aliquots, which were kept reaction, and the regeneration of THF from 5-methyl THF is a frozen at À701C until assayed. vitamin B12-dependent reaction. The plasma concentration Serum homocysteine analyses were conducted using a of these vitamins in pregnant women is known to decrease modification of a high-performance liquid chromatography (Bruinse & van den Berg, 1995) and such decreases could (HPLC)-fluorescence detection method by Araki and Sako adversely affect homocysteine metabolism during preg- (1987). The interassay coefficient of variance (CV) for nancy. homocysteine was 4.9%. Serum levels of flavin adenine In addition, genetic polymorphism of the enzyme dinucleotide (FAD), flavin mononucleotide (FMN), and MTHFR, which catalyzes the irreversible conversion of riboflavin were measured by the HPLC-UV detection 5,10-methylene THF to 5-methyl THF in the folate cycle, is method of Botticher and Botticher (1987). The CVs of the known to influence homocysteine metabolism resulting in FAD, FMN, and riboflavin assay were 5.8, 6.9, and 4.1%, hyperhomocysteinemia (Jacques et al, 1996). The homozyg- respectively. Serum pyridoxal-5-phosphate (PLP) was ana- osity for the C677T variant MTHFR allele causes higher lyzed with a reagent kit for HPLC analysis for vitamin B6 fasting plasma homocysteine concentrations and lower (Chromsystems, Mu¨nchen, Germany). The CV of the PLP folate status than heterozygous or wild-type groups assay was 3.3%. Serum vitamin B12 and folate concentrations (Brattstrom et al, 1998). A curve of steeper slope in subjects were determined using a radioimmunoassay kit (Diagnostic with T/T than in those of C/C genotype has been reported Products Corporation, Los Angeles, CA, USA). The CVs for for an inverse relation between plasma homocysteine folate and vitamin B12 analyses were 4.0 and 4.4%, and folate (van der Put et al, 1995; Nelen et al, 1998) or respectively. vitamin B12 (Jacques et al, 1996; Woodside et al, 1998; Moriyama et al, 2002). Several studies have compared the relation between the MTHFR mutation analysis MTHFR polymorphism, plasma homocysteine levels, and the DNA was extracted from the whole blood using an Aquapure B vitamin status in pregnant women with pregnancy Genomic DNA blood kit (Biorad Pacific Ltd., Kowloon, Hong complications (Goddijn-Wessel et al, 1996; Leeda et al, Kong) and stored at À201C for analysis. DNA fragments were 1998). However, few studies have been reported on these amplified from the genomic DNA via the polymerase chain relations and on homocysteine metabolism in normal reaction (PCR). Amplication was carried out in the PCR women with uncomplicated pregnancies. buffer with 0.5 U Taq DNA polymerase (Takara Shuzo Co., The purpose of this paper is to report on our investigation Shiga, Japan). PCR conditions comprised of an initial on the effect of the interaction between the C677T MTHFR denaturation step at 951C for 9 min, followed by 33 cycles polymorphism and serum B vitamins on the homocysteine of 951C for 1 min, 601C for 1 min, and 721C for 1 min, and a concentration in normal pregnant women. final extension step at 721C for 10 min (Frosst et al, 1995). The PCR product was digested by using HinfI (Takara Shuzo Co., Shiga, Japan.) The C to T substitution at nucleotide 677 Method creates an extra HinFI restriction site that cleaves the original Subjects 198-bp PCR fragment into 175- and 23-bp fragments. Size A total of 177 consecutive normal Korean pregnant women fractionation of the PCR products was by electrophoresis on in their 24–28 week of gestation participated in this study. 2.5% agarose-gel containing 0.5 mg/ml ethidium bromide The criteria for normal pregnancy are as follows: no and was visualized using the ultraviolet light. pregnancy complications, no medications, and free of pregravid chronic diseases. The research protocol was approved by the Human Investigation Review Committee Statistical analysis of Ewha Womans University, and informed consent forms Data were presented as mean7s.d. and percentages. All data were obtained from all subjects. of serum measures were log transformed to normalize their distributions and were presented as geometric mean7s.d. After the ANOVA, Duncan’s multiple range test was Data collection and biochemical analyses performed to determine the significance of the differences Dietary habits and food intake data were collected from the in the means of serum vitamins and homocysteine among participants by the 24-h recall method. Intakes of energy and pregnant women with different genotypes of the MTHFR 2 nutrients including vitamin B2, vitamin B6, folate, and gene. A w analysis was used to test the significant differences European Journal of Clinical Nutrition MTHFR polymorphism and serum B vitamins in pregnant women KN Kim et al 12 in the prevalence of hyperhomocysteinemia among the such as income and education level of the subjects were not three genotypic groups. A Spearman’s correlation coefficient significantly different among the three MTHFR genotype analysis was used to evaluate the relation between homo- groups (Table 1). cysteine and B vitamins. Analysis of covariance was The mean concentrations of serum FAD, FMN, riboflavin, performed to compare the slopes of the three regression PLP, folate, and vitamin B12 were not different among the lines between serum homocysteine and folate by MTHFR carriers of an MTHFR mutant allele (T/T or C/T genotype) genotypes.